Selective enzymatic amidation of c-terminal esters or acids of peptides

ABSTRACT

The present invention relates to a process for the amidation of C-terminal esters or acids of peptide substrates in solution-phase synthesis of peptides, comprising amidating one or more peptide substrates comprising C-terminal esters or acids using the protease subtilisin in any suitable form in the presence of an ammonium salt derived from an acid having a pKa above 0. 
     This process is useful in the production of protected or unprotected peptides.

This application claims the benefit of U.S. Provisional PatentApplication No. 60/945,982, filed Jun. 25, 2007.

The invention relates to a process for selective enzymatic amidation ofC-terminal esters or acids of peptide substrates in solution-phasesynthesis of peptides.

Many biologically active peptides contain a C-terminal primary amidefunction, e.g. gonadorelin, oxytocin, and arginine vasopressin. Thereare several strategies for the synthesis of peptide amides in solution,which can be roughly divided into four different categories.

In the most straightforward approach, synthesis is started from the freeC-terminal amino acyl amide, maintaining the amide in its free formduring the entire synthesis. Although the most obvious approach on firstexamination, syntheses in solution in the presence of a free amidefunction on the growing peptide chain are often fraught with serioussolubility problems due to hydrogen bonding. Moreover, for peptidesynthesis on a manufacturing scale, the DioRaSSP® method is particularlypreferred (EP-A-1,291,356; U.S. Pat. No. 6,864,357; Eggen I. et al.,Org. Process Res. Dev. 2005, 9, 98-101; Eggen I. et al., J. PeptideSci., 2005, 11, 633-641). In this method, the growing peptide chain ismore or less anchored in a permanent organic phase. However, when usinga free amide function, there is a significant risk of losing asubstantial portion of the growing peptide during aqueous washings,especially in the earlier cycles of the synthesis.

In order to circumvent the aforementioned solubility problems, the amidefunction may be protected during the synthesis, hence starting from theC-terminally protected amino acyl amide. This approach also ensures theanchoring of the growing peptide in the organic phase. Protecting groupsfor the amide function may be selected from benzyl (VVeygand, F. et al.,Chem. Ber., 1968, 101, 3623-3641), benzhydryl (Sakakibara, S. et al.,Bull. Chem. Soc. Jpn., 1967, 40, 2164-2167), triphenylmethyl (Sieber, P.et al., B., Tetrahedron Lett., 1991, 32, 739-742), xanthenyl(Shimonishi, Y. et al., Bull. Chem. Soc. Jpn., 1962, 35, 1966-1970), andcyclopropylmethyl type moieties (Carpino, L. A. et al., J. Org. Chem.,1995, 60, 7718-7719), whose lability towards acid is determined by thesubstituents attached to this moiety. A general problem is the pooraccessibility and commercial availability of the starting amino acidderivatives, especially if these contain protected side chains.Moreover, cleavage of the protecting group from the amide function underacidic conditions may result in the formation of alkylated analogues,depending on the actual sequence of the peptide, the conditions fordeprotection and the nature of the protecting group.

A third option for the synthesis towards peptides with a C-terminalamide function is to start from amino acyl esters, which allow selectivecleavage towards the free carboxylic function in the presence ofacid-labile protecting groups on the side chains of the peptide. Thefree carboxylic function is subsequently activated to allow reactionwith ammonia c.q. ammonium. Esters within the scope of this approachinclude 9-fluorenylmethyl (Fm) (Cunningham, B. R. et al., TetrahedronLett., 1994, 35, 9517-9520) and 2-(4-nitrophenylsulfonyl)ethyl (Nse)esters (Carreno, C. et al., J. Peptide Res., 2000, 56, 63-69) which arecleaved under basic conditions, 2-(trimethylsilyl)ethyl (Tmse) esterswhich are cleaved by fluoridolysis (Sieber, P., Helv. Chim. Acta, 1977,60, 2711-2716), and 2,4-dimethoxybenzyl (Dmb) esters which are cleavedby mild acidolysis (McMurray, J. S., Tetrahedron Lett., 1991, 32,7679-7682). The main problem with this approach is that it comprises twopotential racemization-inducing steps of the C-terminal amino acidderivative, i.e. the esterification step and the actual amidation step.Moreover, the cleavage step may lead to side reactions within thepeptide sequence, such as conversion of internal Asp(OBu^(t)) residuesto the succinimide under basic (Mergler, M. et al., J. Pept. Sci., 2003,9, 36-46) or fluoridolytic conditions. Finally, the ester functionitself may not be completely stable during the assembly of the peptidesequence; e.g., benzyl-type esters (Fm and Dmb) are cleaved duringhydrogenolysis, thus ruling out the use of the benzyloxycarbonyl (Z)group for temporary protection of the amino function.

The last option for the synthesis towards peptides with a C-terminalamide function is to start from simple primary amino acyl esters, suchas methyl, ethyl or benzyl esters, which are at a later stage convertedto the amides via direct ammonolysis. These are in general widelyavailable and easily accessible starting compounds. However, thisapproach is limited by the fact that the ammonolytic conditions alsoinduce side reactions within the peptide sequence, such as racemizationand conversion of internal Asp(OBu^(t)) residues to the succinimide.

It may be concluded that the use of simple primary amino acyl esters asthe starting materials in the synthesis towards peptides with aC-terminal amide function is preferred, if the chemical ammonolysis canbe replaced by a mild selective method for the amidation.

Enzymatic methods have gained interest in peptide synthesis during thepast couple of years. Enzymes often show high chemo-, regio- andstereoselectivity. Furthermore, enzymes usually operate under very mildconditions, at neutral pH values and at temperatures of 20-50° C. Thus,under such conditions, side reactions can be circumvented.

In mammals several amidating enzymes are expressed, includingbifunctional Peptidylglycine α-Amidating Monooxygenase (PAM) and themonofunctional enzymes Peptidyl α-Hydroxylating Monooxygenase (PHM) andPeptidyl Amidoglycolate Lyase (PAL) (Kulathila, R. et al., Nat. Prod.Rep., 1999, 16, 145-154). These enzymes need a glycine at the C-terminusand are therefore not generally applicable. Furthermore, these isolatedenzymes are very costly and have found no application in the laboratory({hacek over (C)}e{hacek over (r)}ovský, V. et al, Biotechnol. Appl.Biochem., 2001, 33, 183-187). {hacek over (C)}e{hacek over (r)}ovský etal. reported ({hacek over (C)}e{hacek over (r)}ovskcý, V. et al., Angew.Chem. Int. Ed., 1998, 37, 1885-1887) the application of an enzymeisolated from orange peel in the amidation of the free acid of aC-terminal peptide. Despite some optimization yields never exceeded 35%.Furthermore, in another publication the yields seemed to be largelydependant on the substrate used ({hacek over (C)}e{hacek over (r)}ovský,V., Kula, M. R., Biotechnol. Appl. Biochem., 2001, 33, 183-187).Finally, the {hacek over (C)}e{hacek over (r)}ovský method is based onconversion of peptide with a free C-terminal acid to the correspondingamide. Since solution-phase synthesis usually yields a C-terminal ester,an additional synthesis step is required to deprotect the esterfunction. Accordingly, it will be very difficult to apply this orangepeel enzyme in an industrial process.

The use of lipases has shown a wide spread application in organicchemistry. Several publications report the use of Candida antarcticalipase in the amidation of organic compounds, resulting in substitutedand non-substituted amides (Reyes-Duarte, D. et al., Biotech Lett.,2002, 24, 2057-2061; Sánchez, V. M. et al., J. Org. Chem. 1999, 64,1464-1470; Torre, O. et al., Adv. Synth. Catal. 2005, 347, 1007-1014;Maugard, T. et al., Tetrahedron, 1997, 53, 14, 5185-5194; Zoete de, M.C. et al., Sheldon, R. A. Journal Molecular Catalysis B: Enzymatic,1996, 1, 109-113; Litjens, M. J. J. et al., Tetrahedron, 1999, 55,12411-12418). The application of this enzyme in peptide chemistryhowever remains to be reported.

Finally, Chen S-T. et al., Synthesis, 1993, 858-860, reports theenzymatic amidation of esters of amino acids and C-terminal peptideesters with Alcalase (free subtilisin) in the presence of ammoniumchloride/triethyl amine at pH 10.6 and higher. Reported yields arebetween 50 and 70%. Chen reports in a table the amidation of a dipeptidewith a 68% conversion in 12 hours. The amidation of a tripeptide is alsoreported. However, the conversion is not listed. It may also bementioned here that it is highly undesired in solution-phase synthesisof peptide to use such high pH as this might result in the destructionof the peptide. Therefore, mild conditions, i.e. a pH of 10 or lower,are preferred.

Therefore, despite these positive developments enzymatic amidation ofthe C-terminal ester of a peptide by means of a commercially availableenzyme with high yields remains a daunting challenge hitherto.

A new process has now been found for the amidation of C-terminal estersor acids of peptide substrates in solution-phase synthesis of peptides,comprising amidating one or more peptide substrates comprisingC-terminal esters or acids using the protease subtilisin in any suitableform in the presence of an ammonium salt derived from an acid having apKa above 0.

Surprisingly, it has been found that with the process of the presentinvention high yields of the amidated product can be obtained, whileendopeptidase activity is substantially suppressed.

With C-terminal acids of peptide substrates is meant the free C-terminalacid of a peptide.

Preferred are the C-terminal esters of peptide substrates. Sincesolution-phase synthesis usually yields a C-terminal ester, anadditional synthesis step is required to deprotect the ester function.Furthermore, the amidation of the free C-terminal acid of a peptide ismuch slower than the amidation of the C-terminal ester.

The esters of the C-terminal esters of the peptide substrate may beselected from the group of C₁₋₁₂ (ar)alkyl esters, e.g. methyl, ethyl,propyl, butyl, and benzyl esters, in particular primary C₁₋₁₂ (ar)alkylesters, preferably primary C₁₋₄ alkyl esters, more preferably ethyl andmethyl esters. The C-terminal methyl ester of the peptide substrate isthe most preferred embodiment.

The ammonium salt is derived from an acid having a pKa above 0,preferably from an acid having a pKa above 3.5, most preferably, havinga pKa between 3.5 and 7. Examples include diammonium hydrogen phosphate,ammonium dihydrogen phosphate, ammonium fluoride, or ammonium sulphitehydrate.

Alternatively, the ammonium salt may have the following chemicalstructure (I):

wherein

R1 is selected from the group of hydrogen, C₁₋₁₂ (ar)alkyl, C₆₋₁₂ aryl,—N(R2)₂, —OH, and R3-O⁻NH₄ ⁺,

R2 is selected from the group of hydrogen and/or C₁₋₄ alkyl, and

R3 is a bond, a carbonyl group, or a C₁₋₄ alkyl carbonyl group,optionally substituted with one or more hydroxyl groups and/or —COO⁻NH₄⁺, optionally in hydrated form.

Examples of ammonium salts according to the above-mentioned formulainclude ammonium carbamate, ammonium acetate, ammonium tartrate,ammonium benzoate, ammonium citrate, ammonium formate, ammonium oxalatemonohydrate, ammonium carbonate, ammonium bicarbonate, and mixturesthereof.

Preferably, R1 is selected from the group of C₁₋₁₂ (ar)alkyl, C₆₋₁₂aryl, —NH₂, —OH, and —O⁻NH₄ ⁺.

Preferred embodiments are selected from ammonium carbamate, ammoniumcarbonate, ammonium bicarbonate, ammonium acetate, ammonium benzoate,and mixtures thereof.

Ammonium carbamate is the most preferred embodiment.

It is understood that ammonium carbamate cannot be derived in practicefrom carbamic acid in view of the fact that carbamic acid as such is anunstable compound and thus therefore does not exist. However, for thesake of the present invention it is defined that ammonium carbamate isan ammonium salt derived from carbamic acid, having a pKa between 4.2and 7 (Masuda K. et al., Tetrahedron, 2005, 61, 213-229).

The protease subtilisin (EC 3.4.21.62) may be used in the process of theinvention in any form, thus it may be used in soluble and/orcrystallized form, but also in immobilized form or other insoluble form,e.g. in the form of cross-linked enzyme aggregates (CLEA) orcross-linked enzyme crystals (CLEC).

The new process of this invention may conveniently be used in theproduction of protected or unprotected peptides.

Preferably, the C-terminal ester or acid of the peptide substratescomprises a C-terminal acyl residue which is an α-amino acyl residuefrom natural or synthetic origin. The C-terminal α-amino acyl residuemay be protected or unprotected at the side chain. In particularpreferred are C-terminal α-amino acyl residues selected from Ala,protected Cys, protected Asp, protected Glu, Phe, Gly, H is, (protected)Lys, Leu, Met, Asn, Gln, (protected) Arg, (protected) Ser, Thr, Val,(protected) Trp and (protected) Tyr, wherein the brackets around theword “protected” mean that the residue can be present in both side-chainprotected and unprotected form. The three-letter code for amino acids isused here according to IUPAC nomenclature (IUPAC-IUB Commission (1985)J. Biol. Chem. 260, 14-42).

In a further preferred embodiment, protected or unprotected peptidesubstrates used in the process of this invention are prepared accordingto DioRaSSP®. Thus, a peptide substrate comprising a C-terminal ester oracid is preferably prepared according to this process for rapid solutionsynthesis of a peptide in an organic solvent or a mixture of organicsolvents, the process comprising repetitive cycles of steps (a)-(d):

(a) a coupling step, using an excess of an activated carboxyliccomponent to acylate an amino component,

(b) a quenching step in which a scavenger is used to remove residualactivated carboxylic functions, wherein the scavenger may also be usedfor deprotection of the growing peptide,

(c) one or more aqueous extractions and optionally, (d) a separatedeprotection step, followed by one or more aqueous extractions,

whereby in at least one cycle in process step b an amine comprising afree anion or a latent anion is used as a scavenger of residualactivated carboxylic functions. The amine is preferably benzylβ-alaninate or a salt thereof.

The molar ratio of ammonium salt to peptide substrate may range from 2:1to 20:1, preferably 5:1 to 12:1, more preferably 6:1 to 10:1.

The amidation of the process of the present invention may be performedin one or more organic solvents. Polar organic solvents are preferred,and in particular the organic solvent is selected fromN,N-dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dioxane,N,N-dimethylacetamide (DMA), dichloromethane (DCM), tetrahydrofuran(THF), acetonitrile, tert-butanol, tert-amyl alcohol, dichloroethane(DCE), tert-butyl methyl ether (MTBE), and mixtures thereof. Preferredare mixtures of tert-butanol and DMF, tert-butanol and NMP, tert-amylalcohol and DMF, or tert-amyl alcohol and NMP.

These mixtures may be used in a ratio of 40:60 to 95:5 (v/v), preferably60:40 to 90.10 (v/v), more preferably in a ratio of 82.5:17.5 (v/v).

Preferably, a small amount of water is present in the reaction mixture.It has been found that in a fully anhydrous “dry” system the enzyme isinactivated due to lack of water. However, when the water concentrationis too high in the reaction mixture undesired ester hydrolysis occurs toa significant extent (i.e. formation of a C-terminal free acid) at thecost of amidation. Accordingly, the percentage of water in the mixturemay range from 0.0001 to 5% (v/v), preferably from 0.01 to 2% (v/v). Itis especially useful when the percentage of water in the mixture isabout 0.1 to 1% (v/v).

Water may be added on purpose or by introducing water-containingreagents. For example, when cross-linked enzyme aggregates (CLEA's) areused, water is introduced automatically in the reaction mixture due toits presence in CLEA's.

Introduction of water in the reaction mixture may be carried out in theform of an aqueous buffer. Suitable buffers may be selected from bufferswhich are generally used for transformations using proteolytic enzymes.In particular, the aqueous buffer is a phosphate, borate or2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) buffer.

The pH at which the reaction is performed may be selected from the rangeof 5.5-10, preferably 5.5-8.5, more preferably 6-8, and most preferablythe pH is 7.

Reaction temperatures for the amidation may suitably be selected in therange of 15-60° C., in particular 20 to 40° C. Preferred is a reactiontemperature of 30° C.

The amount of enzyme may suitably be selected in the range of 1 to 50wt. % of enzyme related to the peptide substrate, preferably 5 to 10 wt.%.

In a further embodiment of the invention, the amidation is performed bystepwise adding portions of the protease subtilisin (in any suitableform) to the reaction mixture comprising one or more peptide substratescomprising C-terminal esters or acids.

In an alternative embodiment of the invention, the amidation isperformed by stepwise adding portions of the ammonium salt to thereaction mixture comprising one or more peptide substrates comprisingC-terminal esters or acids.

The peptide substrates, of which the C-terminal esters or acids areamidated in the process of the invention, may carry protecting groups inother parts of their peptide sequence.

The amidation can be carried out with one peptide substrate as well as amixture thereof. Such a mixture may comprise peptide substrates havingonly C-terminal esters, peptide substrates having only free C-terminalacids, or peptide substrates having both C-terminal esters and freeC-terminal acids.

A suitable process according to the present invention is as follows.

To a solution of a C-terminal ester or acid of a peptide substrate in asuitable organic solvent (or mixture of organic solvents) ammonium saltis added. The reaction mixture is incubated above ambient and thereaction is started by the addition of an enzyme catalyst. The enzymecan be added in solution (e.g. Subtilisin A in phosphate buffer), insuspension or as solid material (e.g. native enzyme, immobilised enzymeor cross-linked enzyme). When substrate conversion has reached a desiredlevel, e.g. higher than 90%, the peptide may be isolated according tothe knowledge of the skilled person.

The term ‘substrate’ herein means an entity which is converted to aproduct by the protease subtilisin in any form. This product may be afinal peptide product whereby if present only the protected functionalside chains have still to be deprotected. Alternatively, this productmay also be a peptide fragment which subsequently is reacted with otherpeptide fragments in a convergent synthesis to obtain a longer peptidewith the required final number of amino acids.

A person skilled in the art can easily identify suitable substrates, forinstance by performing a simple test amidation of a selected peptidecomprising a C-terminal ester functionality or a free C-terminal acidunder suitable conditions as described herein before and followingconversion e.g. by HPLC techniques.

It is established that the process is very suitable to prepare amides ofshort peptides comprising up to 5 amino acids. Furthermore, a skilledperson will also be able to prepare longer peptides. Peptides may alsobe prepared with D-amino acids.

The term ‘protected’ means that the functional groups (within thepeptide) are protected with suitable protecting groups. A person skilledin the art will know which type of protection to select for which typeof functional group. For example, amine functions present in thecompounds may be protected during the synthetic procedure by anN-protecting group, which means a group commonly used in peptidechemistry for the protection of an α-amino group, like thetert-butyloxycarbonyl (Bac) group, the benzyloxycarbonyl (Z) group, orthe 9-fluorenylmethyloxycarbonyl (Fmoc) group. Overviews of aminoprotecting groups and methods for their removal is given in Geiger R.and König W. (1981) in Peptides: Analysis, Synthesis, Biology, Vol 3,Gross E. and Meienhofer, J., eds, Academic Press, New York, pp. 1-99,and Peptides: Chemistry and Biology, Sewald N. and Jakubke H.-D., eds,Wiley-VCH, Weinheim, 2002, pp. 143-154. Functions of the tert-butyl typeor functions of similar lability are preferred for the protection ofother functional groups on the side chains; these include—but are notlimited to—tert-butyl (Bu^(t)) for the protection of the Asp, Glu, Ser,Thr and Tyr side chains, tert-butoxycarbonyl (Boc) for the protection ofthe Lys and Trp side chains, trityl (Trt) for the protection of the Asn,Gln and His side chains and 2,2,5,7,8-pentamethylchromane-6-sulfonyl(Pmc) or 2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Pbf) forthe protection of the Arg side chain [Barany, G. and Merrifield, R. B.(1980) in: ‘The Peptides’, vol. 2 (Gross, E. and Meienhofer, J., eds.)Academic Press, New York, pp. 1-284; for Trp(Boc): Franzén, H. et al.(1984) J. Chem. Soc., Chem. Commun., 1699-1700; for Asn(Trt) andGln(Trt): Sieber, P. et al. (1991) Tetrahedron Lett. 32, 739-742; forHis(Trt): Sieber, P. et al. (1987) Tetrahedron Lett. 28, 6031-6034; forPmc: Ramage, R. et al. (1987) Tetrahedron Lett. 28, 2287-2290; for Pbf:Carpino, L. A. et al. (1993) Tetrahedron Lett. 34, 7829-7832].

The invention is further illustrated by the following examples, which isnot to be interpreted as a limitation of this invention.

EXAMPLES

The peptides have been produced according to conventional solution phasemethods for peptide synthesis.

The free enzyme Subtilisin A was purchased from Novozymes unlessotherwise stated. Alcalase CLEA, Savinase CLEA, and CAL-B CLEA wereobtained from CLEA Technologies B. V., Delft, The Netherlands. All otherenzymes were purchased from Sigma Aldrich.

-   P—NH₂=Z-peptide-NH₂, e.g. Z-Val-Phe-NH₂-   P—OH=Z-peptide-OH, e.g. Z-Val-Phe-OH-   P—OMe=Z-peptide-OMe, e.g. Z-Val-Phe-OMe-   Bu^(t)OH=tert-butanol-   Am^(t)OH=tert-amyl alcohol-   DMF=N,N-dimethylformamide-   NMP=N-methyl-2-pyrrolidone-   DMA=N,N-dimethylacetamide-   DCM=dichloromethane-   THF=tetrahydrofuran-   DCE=dichloroethane

Example 1 and Comparative Examples A-G General Procedure for Screeningthe Effect of Enzyme

A stock solution of Z-Ala-Phe-OMe in DMF (200 mM) was prepared. 0.5 mmolof ammonium carbamate was added to 0.25 ml of the dipeptide stocksolution. Next, 0.625 ml DMF and 4.125 ml Bu^(t)OH was added. Dependingon the enzyme used 10 μl phosphate buffer 0.1 M pH 7.0 was added. Thereaction mixture was incubated at 30° C. and the reaction was started bythe addition of approximately 20 mg of enzyme. A sample (2 ml) was takenafter two hours. 2 ml of acetonitrile was added and the mixture wascentrifuged. The supernatants were analyzed by HPLC. The results areshown in Table 1.

TABLE 1 Z-Ala- Z-Ala- Z-Ala- H₂O/Bu^(t)OH/ Phe-NH₂ Phe-OH Phe-OMe Ex.DMF a/a % a/a % a/a % Enzyme 1 0.2/82.5/17.5 86.2 13.4 0.4 Alcalase CLEAA 0.2/82.5/17.5 5 2.6 92.4 Savinase CLEA B 0.2/82.5/17.5 0 0 100 CAL-BCLEA

As is seen from the results in Table 1 Candida Antartica Lipase-B CLEA(CAL-B CLEA) and Savinase-CLEA are not active.

In similar protocols as described above Carboxypeptidase A (Comp. Ex.C), Lipase CC (Candida Cylindracea) (Comp. Ex. D), Lipase CR (CandidaRugosa) (Comp. Ex. E), Esterase HL (hog liver) (Comp. Ex. F), and LipaseCA (AR) (CAL-B immobilized on acrylic resin) (Comp. Ex. G) were notactive. The reaction mixtures were also analyzed after 16 hours,providing similar results.

Examples 2 and 3 and Comparative Example H General Procedure forScreening the Effect of Ammonium Source

A stock solution of Z-Ala-Phe-OMe in DMF (50 mM) was prepared. 0.5 mmolof the ammonium source was added to 1 ml of the dipeptide stocksolution. 4 ml Bu^(t)OH was added. 50 μl phosphate buffer 0.1 M pH 7.6was added. The reaction mixture was incubated at 40° C. and the reactionwas started by the addition of 5 mg of Alcalase-CLEA. A sample (2 ml)was taken after two hours. 2 ml of acetonitrile was added and themixture was centrifuged. The supernatants were analyzed by HPLC. Theresults are shown in Table 2.

TABLE 2 Composition reaction mixture Ammonium Z-Ala-Phe- Z-Ala-Phe-Z-Ala-Phe- Ex. source NH₂ a/a % OH a/a % OMe a/a % 2 (NH₄)₂CO₃ 11.8 6.881.3 3 NH₂CO₂NH₄ 16.1 8.7 75.2 H NH₄Cl 0.4 7.2 92.4

Example 4 Procedure for Enzymatic Amidation of a Tripeptide

A stock solution of Z-Ala-Phe-Ala-OMe in DMF (200 mM) was prepared. 0.5mmol of NH₂CO₂NH₄ was added to 0.25 ml of the tripeptide stock solution.0.625 ml DMF and 4.125 ml Bu^(t)OH was added. The reaction mixture wasincubated at 30° C. and the reaction was started by the addition of 20mg of Alcalase-CLEA. A sample (2 ml) was taken after two hours and afterfour hours. 2 ml of acetonitrile was added and the mixture wascentrifuged. The supernatants were analyzed by HPLC. The results arepresented in Table 3.

TABLE 3 Time Z-Ala-Phe-Ala- Z-Ala-Phe-Ala- Z-Ala-Phe-Ala- (hours) NH₂a/a % OH a/a % OMe a/a % 2 69.7 10.2 18.3 4 79.0 10.6 8.9

Example 5 Effect of “Dry” Environment on the Amidation of a Dipeptidewith Free Subtilisin

All solvents were dried with mol sieves prior to the experiment.

A stock solution of Z-Ala-Phe-OMe in DMF (200 mM) was prepared. 0.5 mmolof ammonium carbamate was added to 0.25 ml of the dipeptide stocksolution. 0.625 ml DMF and 4.125 ml Bu^(t)OH was added. The reactionmixture was incubated at 30° C. and the reaction was started by theaddition of 1 mg of enzyme Subtilisin A. A sample (2 ml) was taken aftertwo hours. 2 ml of acetonitrile was added and the mixture wascentrifuged. The supernatants were analyzed by HPLC. The results areshown in Table 4.

TABLE 4 Z-Ala-Phe- Z-Ala-Phe- Z-Ala-Phe- Water/Bu^(t)OH/DMF NH₂ a/a % OHa/a % OMe a/a % 0/82.5/17.5 1.7 0.4 97.9

Examples 6 to 14 Effect of Additional Ammonium Carbamate and/or Enzymeon the Amidation of a Dipeptide when Subtilisin A is Used

A stock solution of Z-Ala-Phe-OMe in DMF (200 mM) was prepared. 0.5 mmolof ammonium carbamate was added to 0.25 ml of the dipeptide stocksolution. DMF and Bu^(t)OH was added corresponding to the ratio given inTable 5, for a total reaction volume of 5 ml. The reaction mixture wasincubated at 30° C. and the reaction was started by the addition of 10μl of enzyme solution (20 mg Subtilisin A in 200 μl phosphate buffer 0.1M pH 7.0) (Initial conditions: 10 mM Z-Ala-Phe-OMe, 100 mM NH₂COONH₄, pH7, 30° C.). A sample (2 ml) was taken after 1.5 hours for experiments 6,9, and 12, and after 3 hours for experiments 7, 8, 10, 11, 13, and 14. 2ml of acetonitrile was added and the mixture was centrifuged. Thesupernatants were analyzed by HPLC. The results are shown in Table 5.

TABLE 5 Z-Ala- Z-Ala- Z-Ala- H₂O/Bu^(t)OH/ Phe-NH₂ Phe-OH Phe-OMe Ex.DMF a/a % a/a % a/a % Comments 6 0.2/82.5/17.5 62.1 11.6 26.2 70.2/82.5/17.5 75 10.7 14.2 extra carbamate (0.5 mmol at 1.5 h) 80.2/82.5/17.5 77.2 12.3 10.6 extra carbamate (0.5 mmol) & enzyme (10 μl)at 1.5 h 9 0.2/70/30 67.3 9.6 23.1 10 0.2/70/30 75.8 9.3 14.9 extracarbamate (0.5 mmol at 1.5 h) 11 0.2/70/30 81.7 10.9 7.5 extra carbamate(0.5 mmol) & enzyme (10 μl) at 1.5 h 12 0.2/40/30 53.6 7.2 39.1 130.2/40/60 56.5 6.3 37.2 extra carbamate (0.5 mmol at 1.5 h) 14 0.2/40/6068.6 8.4 23 extra carbamate (0.5 mmol) & enzyme (10 μl) at 1.5 h

The results show that addition of extra ammonium carbamate and/or enzymeresults in an increase in substrate conversion and formation of higheramounts of amide.

Examples 15 to 18 Effect of Additional Ammonium Carbamate on theAmidation of a Dipeptide when Alcalase CLEA is Used

A stock solution of Z-Ala-Phe-OMe in DMF (200 mM) was prepared. 0.5 mmolof the ammonium source was added to 0.25 ml of the dipeptide stocksolution. 0.625 ml DMF and 4.125 ml Bu^(t)OH was added. 10 μl phosphatebuffer 0.1 M pH 7.0 was added in experiments 17 and 18. The reactionmixture was incubated at 30° C. and the reaction was started by theaddition of approximately 20 mg of Alcalase-CLEA. A sample (2 ml) wastaken after two hours. 2 ml of acetonitrile was added and the mixturewas centrifuged. The supernatants were analyzed by HPLC. The results areshown in Table 6 (initial conditions: 10 mM Z-Ala-Phe-OMe, 100 mMNH₂COONH₄, pH 7, 30° C.).

TABLE 6 Z-Ala- Z-Ala- Z-Ala- H₂O/Bu^(t)OH/ Phe-NH₂ Phe-OH Phe-OMe Ex.DMF a/a % a/a % a/a % Comments 15 0/82.5/17.5 90.9 8.4 0.7 160/82.5/17.5 91.1 7.8 1.1 extra carbamate (0.05 mmol at 1 h) 170.2/82.5/17.5 84.7 14.4 0.9 18 0.2/82.5/17.5 78.9 20.1 1 extra carbamate(0.05 mmol at 1 h)

The results show that Alcalase CLEA is highly efficient under theconditions tested. 99% of Z-Ala-Phe-OMe was converted. When noadditional buffer was added to the reaction mixture, the productcontained approx. 91% Z-Ala-Phe-NH₂ and 8% free Z-Ala-Phe-OH. Additionof water increases ester hydrolysis yielding the free acid.

Examples 19 and 20 Effect of Additional DMF on the Amidation of aDipeptide when Alcalase CLEA is Used

A stock solution of Z-Ala-Phe-OMe in DMF (200 mM) was prepared. 0.5 mmolof the ammonium source was added to 0.25 ml of the dipeptide stocksolution. DMF and Bu^(t)OH was added corresponding to the ratio given inTable 5, for a total reaction volume of 5 ml. The reaction mixture wasincubated at 30° C. and the reaction was started by the addition ofapproximately 20 mg of Alcalase-CLEA. A sample (2 ml) was taken aftertwo hours. 2 ml of acetonitrile was added and the mixture wascentrifuged. The supernatants were analyzed by HPLC. The results areshown in Table 7 (initial conditions: 10 mM Z-Ala-Phe-OMe, 100 mMNH₂COONH₄, pH 7, 30° C.).

TABLE 7 H₂O/Bu^(t)OH/ Z-Ala-Phe- Z-Ala-Phe- Z-Ala-Phe- Ex. DMF NH₂ a/a %OH a/a % OMe a/a % 19 0/70/30 91.4 7.6 1.1 20 0/60/40 91 7.4 1.6

Examples 21 to 30 General Procedure for Screening Different Substrateswith Alcalase CLEA and Ammonium Carbamate

In a typical experiment, the assay mixture contained approximately 10 mMsubstrate and 100 mM ammonium carbamate in an anhydrous solvent mixtureof 17.5% (vol/vol) DMF in Bu^(t)OH in a total reaction volume of 5 ml.The reaction mixture was thermostated at 30° C. The reaction wasinitiated by the addition of 4 mg/ml of Alcalase-CLEA. Reaction wasincubated at 30° C. for 75 hours. Aliquot samples were taken at 2 h, 4 hand 75 h and the reaction was stopped by the addition of an equal volumeof acetonitrile. Samples were analyzed by HPLC. All experiments wereperformed in duplo.

TABLE 8 Reaction P—NH₂ P—OH P—OMe Ex. Peptide substrate time (h) a/a %a/a % a/a % 21 Z-Val-Phe-OMe 2 77.0 16.4 6.7 4 82.1 17.2 6.7 75 86.713.4 0.0 22 Z-Val-Tyr-OMe 2 41.2 13.1 45.8 4 44.4 20.0 35.7 75 59.3 40.00.8 23 Z-Val-Leu-OMe 2 73.4 19.5 7.2 4 73.5 26.5 0.0 24 Z-Val-Thr-OMe 287.4 7.7 5.0 4 90.8 9.2 0.0 25 Z-Val-Ala-OMe 2 88.9 11.2 0.0 26Z-Val-Met-OMe 2 71.1 28.1 0.9 4 72.8 27.3 0.0 27 Z-Val-Lys(Boc)-OMe 222.9 7.2 70.0 4 33.1 12.2 54.7 75 74.8 24.0 1.3 28 Z-Ala-Phe-OMe 2 91.76.7 1.7 4 92.9 6.7 0.5 29 Z-Ala-Phe-Ala-OMe 2 82.8 8.5 8.8 4 92.2 6.91.0 30 Z-Ala-Phe-D-Ala-OMe 2 6.0 0.3 93.7 4 10.8 0.8 88.5 75 35.4 10.454.3

With the exception of Z-Val-Tyr-OMe, Z-Val-Lys(Boc)-OMe andZ-Ala-Phe-D-Ala-OMe, all substrate peptides were quantitativelyconverted into products, i.e. the corresponding amide peptide and freecarboxylic acid peptide, in 4 h reaction time. At longer reaction times,also the methyl ester peptides ending with Tyr and Lys(Boc) were totallyconverted. Surprisingly, the methyl ester of the tripeptide with aC-terminal D-Ala residue was also converted. The HPLC analysis at 75 hreaction time showed conversion of 54 a/a % of substrate to yield about36 a/a % of the product Z-Ala-Phe-D-Ala-NH₂.

Examples 31 to 45 and Comparative Example I General Procedure forScreening the Effect of Different Ammonium Salts

In a typical experiment, the assay mixture contained approximately 10 mMpeptide ester Z-Ala-Phe-OMe and 100 mM ammonium salt in an anhydroussolvent mixture of 17.5% (vol/vol) DMF in Bu^(t)OH in a total reactionvolume of 5 ml. The reaction mixture was thermostated at 30° C. Thereaction was initiated by the addition of the enzyme (Alcalase-CLEA: 4mg/ml or Subtilisin A (from SigmaAldrich): 1 mg/ml). Reaction wasincubated at 30° C. for 21 hours. Aliquot samples were taken at 2 h, 4 hand 21 h and the reaction was stopped by the addition of an equal volumeof acetonitrile. Samples were analyzed by HPLC. All experiments wereperformed in duplo. The results for Alcalase CLEA are shown in Table 9.The results for Subtilisin A are shown in Table 10.

TABLE 9 (Alcalase CLEA) Z-Ala- Z-Ala- Z-Ala- Ammonium Reaction Phe-NH₂Phe-OH Phe-OMe Ex. salt time h a/a % a/a % a/a % 31 NH₂COONH₄ 2 92.3 7.70 Carbamate 4 92.3 7.7 0 21 91.4 8.6 0 32 CH₃COONH₄ 2 83.4 12.5 4.1Acetate 4 87.2 12.3 0.5 21 91.2 8.8 0 33 (NH₄)₂CO₃ 2 88.1 11.9 0.0Carbonate 4 88.8 11.2 0.0 21 90.8 9.2 0 34 NH₄HCO₃ 2 84.7 14.9 0.4Bicarbonate 4 85.7 14.3 0 21 88.3 11.7 0 35 C₆H₅COONH₄ 2 61.1 30.4 8.5Benzoate 4 68.7 29.8 1.5 21 87.7 12.3 0 36 HCOONH₄ 2 31.6 19.8 48.5Formate 4 41.9 26.6 31.5 21 69.5 30.5 0 37 NH₄F 2 26.9 15.1 57.9Fluoride 4 41.2 20.5 38.2 21 71.0 27.0 1.9 38 (NH₄)₃C₆H₅O₇ 2 5.6 47.946.4 Citrate 4 12.6 61.0 26.4 21 77.6 22.4 0 39 (NH₄)₂C₂O₄•H₂O 2 3.748.2 48.1 Oxalate 4 10.5 62.9 26.6 monohydrate 21 68.1 31.9 0 40(NH₄)₂C₄H₄O₆ 2 6.3 37.7 55.9 Tartrate 4 17.6 50.8 31.6 21 68.2 31.8 0 41(NH₄)₂SO₃•H₂O 2 2.9 6.1 91.0 Sulphite 4 7.2 12.8 80.0 hydrate 21 60.739.3 0 I NH₄Cl 2 6.8 38.8 54.4 Chloride 4 14.8 52.9 32.3 21 68.5 31.5 0

Ammonium carbamate, ammonium acetate, ammonium carbonate, ammoniumbicarbonate, and ammonium benzoate show excellent performance.

TABLE 10 (Subtilisin A) Z-Ala- Z-Ala- Z-Ala- Ammonium Reaction Phe-NH₂Phe-OH Phe-OMe Ex. source time h a/a % a/a % a/a % 42 NH₂COONH₄ 2 36.94.8 48.4 Carbamate 4 60.2 8.2 31.6 21 86.8 12.9 0.3 43 CH₃COONH₄ 2 42.47.9 49.7 Acetate 4 58.6 11.6 29.8 21 82.1 17.1 0.8 44 NH₄HCO₃ 2 27.2 9.862.9 Bicarbonate 4 46.6 16.1 37.3 21 76.0 23.7 0.3 45 (NH₄)₂CO₃ 2 42.47.9 49.7 Carbonate 4 53.1 12.0 34.9 21 80.9 8.7 0.4

Table 10 shows the results of the conversion of Z-Ala-Phe-OMe into amidewith Subtilisin A, with ammonium carbamate, ammonium acetate, ammoniumbicarbonate and ammonium carbonate as source of ammonia. High substrateconversion is obtained after 21 h reaction for the selected salts.Highest yield (i.e. 86%) of the amide Z-Ala-Phe-NH₂ was obtained forammonium carbamate at a reaction time of 21 h. More product of the esterhydrolysis is obtained for free Subtilisin A as compared toAlcalase-CLEA.

Examples 46 to 53 General Procedure for Screening Different Substrateswith Subtilisin a and Ammonium Carbamate

In a typical experiment, the assay mixture contained approximately 10 mMpeptide ester and 100 mM ammonium carbamate in an anhydrous solventmixture of 17.5% (vol/vol) DMF in Bu^(t)OH in a total reaction volume of5 ml. The reaction mixture was thermostated at 30° C. The reaction wasinitiated by the addition of 1 mg/ml of Subtilisin A (from SigmaAldrich). Reaction was incubated at 30° C. for 21 hours. Aliquot sampleswere taken at 2 h, 4 h and 21 h and the reaction was stopped by theaddition of an equal volume of acetonitrile. Samples were analyzed byHPLC. All experiments were performed in duplicate. Results are given inTable 11.

TABLE 11 Reaction P—NH₂ P—OH P—OMe Ex. Peptide substrate time, h a/a %a/a % a/a % 46 Z-Val-Phe-OMe 2 14.8 5.6 79.6 4 26.9 10.7 62.4 21 61.029.1 9.9 47 Z-Val-Tyr-OMe 2 5.8 3.0 91.2 4 11.1 5.6 83.3 21 29.6 18.352.1 48 Z-Val-Leu-OMe 2 14.3 6.9 78.8 4 25.9 12.1 62.0 21 58.0 33.7 8.349 Z-Val-Thr-OMe 2 18.2 2.3 79.4 4 34.4 4.5 61.1 21 76.7 13.7 9.6 50Z-Val-Ala-OMe 2 50.1 10.0 39.9 4 70.0 14.4 15.6 21 82.1 17.9 0.0 51Z-Val-Met-OMe 2 27.5 7.8 64.7 4 42.3 15.6 42.2 21 55.7 44.3 0.0 52Z-Val-Lys(Boc)-OMe 2 1.6 0.0 98.4 4 2.9 1.2 95.9 21 10.5 6.7 82.7 53Z-Ala-Phe-OMe 2 36.9 4.8 58.4 4 60.2 8.2 31.6 21 86.8 12.9 0.3

The specificity of Alcalase CLEA for various terminal amino acids wasreported in Examples 21 to 30. Here, the results are reported obtainedfor testing the substrate specificity of soluble subtilisin. All peptidesubstrates were transformed into amides, but with lower amide yieldsthan in the case of Alcalase-CLEA. Best substrates were Z-Ala-Phe-OMe(86.8% amide after 21 h) and Z-Val-Ala-OMe (82% amide formed after 21h), while Z-Val-Lys(Boc)-OMe was the worse substrate. Extensive esterhydrolysis was observed. It appears that Alcalase CLEA and Subtilisin Ahave slightly different substrate specificity.

Examples 54 to 68 General Procedure for Screening Different PolarOrganic Solvents

In a typical experiment, the assay mixture contained approximately 10 mMZ-Ala-Phe-OMe and 100 mM ammonium carbamate in an anhydrous solventmixture containing 82.5% dry Bu^(t)OH or Am^(t)OH and 17.5% (vol/vol) ofa co-solvent. The total reaction volume was 5 ml. The reaction mixturewas thermostated at 30° C. The reaction was initiated by the addition ofthe enzyme (Alcalase-CLEA: 4 mg/ml or Subtilisin A (Sigma Aldrich): 1mg/ml). Reaction was incubated at 30° C. for 22 hours. Aliquot sampleswere taken at 2 h, 4 h and 22 h and the reaction was stopped by theaddition of an equal volume of acetonitrile. Samples were analyzed byHPLC. All experiments were performed in duplicate. The results forAlcalase CLEA are shown in Table 12. The results for Subtilisin A areshown in Table 13.

TABLE 12 (Alcalase CLEA) Solvent Z-Ala- Z-Ala- Z-Ala- (82.5:17.5Reaction Phe-NH₂ Phe-OH Phe-OMe Ex. (% vol/vol)) time h a/a % a/a % a/a% 54 Bu^(t)OH/DMF 2 92.0 8.0 0.0 4 92.1 7.9 0.0 22 91.3 8.7 0.0 55Bu^(t)OH/NMP 2 90.0 7.6 2.4 4 91.9 7.8 0.3 22 91.3 8.7 0.0 56Bu^(t)OH/dioxane 2 88.5 8.2 3.2 4 91.3 8.2 0.4 22 92.2 7.8 0.0 57Bu^(t)OH/DMA 2 92.0 7.6 0.5 4 92.2 7.8 0.0 22 90.3 9.7 0.0 58Bu^(t)OH/DCM 2 87.1 10.3 2.6 4 89.1 10.6 0.3 22 91.7 8.3 0.0 59Bu^(t)OH/THF 2 91.5 7.8 0.7 4 92.3 7.7 0.0 22 93.3 6.7 0.0 60Bu^(t)OH/CH₃CN 2 91.5 8.5 0.0 4 91.7 8.3 0.0 22 91.5 8.2 0.0 61Bu^(t)OH/DCE 2 89.7 9.3 1.0 4 90.4 9.6 0.0 22 89.0 10.6 0.3 62Am^(t)OH/DMF* 2 78.0 4.4 17.7 4 89.9 5.5 4.6 22 94 6 0 63 Am^(t)OH/NMP*2 48.1 2.8 49.0 4 65.8 4.5 29.7 22 90.4 8.7 0.9 *Alcalase CLEAconcentration: 1 mg enzyme/ml reaction mixture. In all otherexperiments, the enzyme was used in concentration of 4 mg/ml.

TABLE 13 (Subtilisin A) Solvent Z-Ala- Z-Ala- Z-Ala- (82.5:17.5 ReactionPhe-NH₂ Phe-OH Phe-OMe Ex. (% vol/vol)) time h a/a % a/a % a/a % 64Bu^(t)OH/DMF 2 36.9 4.8 58.4 4 60.2 8.2 31.6 21 86.8 12.9 0.3 65Bu^(t)OH/NMP 2 25.1 3.4 71.5 4 43 6 51 21 83.3 13.5 3.2 66Bu^(t)OH/dioxane 2 9.9 1.4 88.6 4 16.3 2.4 81.3 21 44.6 7.5 47.9 67Bu^(t)OH/THF 2 23.4 3.3 73.3 4 36 5.2 58.8 21 70.3 12.1 17.6 68Am^(t)OH/DMF 2 22.2 3.5 74.3 4 36.9 6.1 57 21 73.4 21.1 5.4

Table 12 shows the results obtained for screening solvent effects whenusing Alcalase CLEA as catalyst. After 2 hours incubation, inexperiments containing solvent mixtures of 82.5 Bu^(t)OH/17.5×(%vol/vol), when high amount of Alcalase CLEA was used (i.e. 4 mgenzyme/ml reaction mixture) total substrate conversion was obtained forall solvent combination used. The reaction mixture contained more than87% amide, and the amide yield increased above 93% at longer reactiontime.

Am^(t)OH proves to be an excellent solvent for the amidation of peptidemethyl esters. When mixtures of Am^(t)OH/DMF (82.5/17.5 vol/vol) wereused as solvent mixture, even at a lower enzyme concentration (AlcalaseCLEA added approximately 1 mg enzyme/ml reaction mixture), high yieldsof amide (i.e. about 80% amide) were obtained after 2 hours ofincubation. At longer reaction time, at total substrate conversion, theproduct mixture contained more than 90, % amide. This suggests thatAm^(t)OH can replace the Bu^(t)OH as main co-solvent in the reactionmixture for enzymatic amidation of methyl esters of peptides. Similartrends were obtained when Subtilisin A was used (Table 13). However,Subtilisin A performs less than Alcalase CLEA in amide synthesis. About85% amide is obtained after 21 hours in 82.5 Bu^(t)OH/17.5 DMF (%vol/vol). Generally, the amount of free peptide resulted from esterhydrolysis is higher, around 12-15%. Binary solvent mixtures containing82.5% Bu^(t)OH or Am^(t)OH in combination with 17.5% of either DMF andNMP seem to be the best solvent mixtures for Subtilisin A in amidesynthesis.

Examples 69 to 82

Since Am^(t)OH seemed to be a very good co-solvent in combination withDMF and NMP for the enzymatic amidation reaction as shown in Examples 55to 69, these solvent mixtures were tested with the same procedure forsome model reactions with Alcalase CLEA and Subtilisin A, for differentpeptide substrates and ammonium salts, respectively. Results are givenin Table 14 for Alcalase CLEA (1 mg/ml) and Table 15 for Subtilisin A(0.2 mg/ml). The conclusion of these experiments is that Am^(t)OH can beused to replace Bu^(t)OH as cosolvent.

TABLE 14 (Alcalase CLEA) Composition of reaction mixture ReactionAmmonium Peptide Solvent % % % Ex. time, h salt Substrate (82.5:17.5)P—NH₂ P—OH P—OMe 69 2 NH₂COONH₄ Z-Ala-Phe-OMe Am^(t)OH/DMF 78.0 4.4 17.74 89.9 5.5 4.6 21 94.0 6.0 0.0 70 2 NH₂COONH₄ Z-Val-Thr-OMe Am^(t)OH/DMF47.9 2.8 49.3 4 64.8 4.6 30.6 21 80.3 14.6 5.1 71 2 NH₂COONH₄Z-Val-Ala-OMe Am^(t)OH/DMF 78.5 8.6 12.9 4 86.2 9.9 4.0 21 87.6 10.6 1.872 2 NH₂COONH₄ Z-Ala-Phe-OMe Am^(t)OH/NMP 48.1 2.8 49.0 4 65.8 4.5 29.721 90.4 8.7 0.9 73 2 NH₂COONH₄ Z-Val-Thr-OMe Am^(t)OH/NMP 39.4 2.0 58.54 57.1 3.7 39.3 21 85.7 10.4 3.9 74 2 NH₂COONH₄ Z-Val-Ala-OMeAm^(t)OH/NMP 60.1 7.7 32.2 4 74.7 10.3 14.9 21 85.3 13.1 1.6 75 2NH₂COONH₄ Z-Ala-Phe-OMe Am^(t)OH/DMF 78 4.4 17.7 4 89.9 5.5 4.6 21 94 60 76 2 (NH₄)₂CO₃ Z-Ala-Phe-OMe Am^(t)OH/DMF 68.7 6.1 25.2 4 83.5 8.1 8.321 90.9 9.1 0 77 2 CH₃COONH₄ Z-Ala-Phe-OMe Am^(t)OH/DMF 39 2.6 58.4 452.8 3.9 43.3 21 86 9.9 4.1

TABLE 15 (Subtilisin A) Composition of reaction mixture ReactionAmmonium Peptide Solvent % % % Ex. time h salt Substrate (82.5:17.5)P—NH₂ P—OH P—OMe 78 2 NH₂COONH₄ Z-Val-Thr-OMe Am^(t)OH/DMF 10.9 1.9 87.34 21.2 3.4 75.4 21 57.0 16.5 26.5 79 2 NH₂COONH₄ Z-Val-Ala-OMeAm^(t)OH/DMF 31.0 8.7 60.3 4 49.7 14.1 36.2 21 73.6 24.2 2.2 80 2NH₂COONH₄ Z-Ala Phe-OMe Am^(t)OH/DMF 22.2 3.5 74.3 4 36.9 6.1 57.0 2173.4 21.1 5.4 81 2 (NH₄)₂CO₃ Z-Ala Phe-OMe Am^(t)OH/DMF 24.9 6.5 68.6 432.0 9.2 58.8 21 69.1 26.5 4.4 82 2 CH₃COONH₄ Z-Ala Phe-OMe Am^(t)OH/DMF30.3 5.8 63.9 4 45.2 9.7 45.1 21 73.7 21.0 5.3

Examples 83 to 91 Optimizing the Amount of Alcalase CLEA in the ReactionMixture

Previous experiments showed that Alcalase CLEA is very efficient in theamide synthesis. Experiments were performed to determine the lowestenzyme concentration that can be used in the reaction, to obtain highproduct yield in a reasonable reaction time.

In a typical experiment, the assay mixture contained approximately 10 mMpeptide substrate and 100 mM ammonium carbamate in an anhydrous solventmixture of 82.5% Bu^(t)OH and 17.5% (vol/vol) of DMF, in a totalreaction volume of 5 ml. The reaction mixture was thermostated at 30° C.The reaction was initiated by the addition of the enzyme (Alcalase-CLEA:0.2 mg/ml, 1 mg/ml and 2 mg/ml, respectively). Reaction was incubated at30° C. for 21 hours. Aliquot samples were taken at 2 h, 4 h and 21 h andthe reaction was stopped by the addition of an equal volume ofacetonitrile. Samples were analyzed by HPLC. All experiments wereperformed in duplicate. The results are listed in Table 16.

TABLE 16 Enzyme Reaction concentration, % % % Ex. time, h mg/ml P—NH2P—OH P—OMe Z-Ala-Phe-OMe 83 2 0.2 33.5 1.2 65.3 4 0.2 51.7 2.4 45.9 210.2 85 6.4 8.6 84 2 1 80.4 3.4 16.2 4 1 92.3 4.2 3.5 21 1 95.1 4.9 0 852 2 91.1 5.4 3.6 4 2 94.5 5.5 0 21 2 94 6 0 Z-Val-Phe-OMe 86 2 0.2 14.51.3 84.1 4 0.2 24.2 2.5 73.3 21 0.2 55.7 11.4 32.9 87 2 1 49.8 5.2 44.94 1 67.8 7.6 24.6 21 1 87.8 11.8 0.4 88 2 2 69.5 9.7 20.8 4 2 81.7 11.76.6 21 2 88.4 11.6 0.0 Z-Val-Thr-OMe 89 2 0.2 56.7 2.6 40.7 4 0.2 76.43.9 19.7 21 0.2 93.5 6.5 0.0 90 2 1.2 76.4 3.9 19.7 4 1.2 76.4 3.9 19.721 1.2 93.5 6.5 0.0 91 2 2 68.8 4.2 27.0 4 2 84.1 5.8 10.0 21 2 90.3 9.70.0

It can be seen that at an Alcalase CLEA concentration of 1 mg/mlreaction mixture, 95% conversion of the substrate Z-Ala-Phe-OMe isobtained after 4 hours incubation. The product mixture contained 92%amide and only 3.5% of the secondary product of the ester hydrolysis.High amide yields were obtained for other peptide substrates, for thislow enzyme concentration, but at longer reaction times. However, theseresults show that the concentration of enzyme, i.e. Alacalse-CLEA, canbe reduced significantly, thus making the process more economical.

Example 92 Amidation of Boc-Pro-Pro-Ala-Phe-Ala-OMe

In a typical experiment, the assay mixture contained approximately 10 mMpeptide substrate and 100 mM ammonium carbamate in an anhydrous solventmixture of 82.5% Bu^(t)OH and 17.5% (vol/vol) of DMF, in a totalreaction volume of 5 ml. The reaction mixture was thermostated at 30° C.The reaction was initiated by the addition of 4 mg/ml Alcalase CLEA.Reaction was incubated at 30° C. for 21 hours. Aliquot samples weretaken at 2 h, 4 h and 21 h and the reaction was stopped by the additionof an equal volume of acetonitrile. Samples were analyzed by HPLC. Theresults are listed in Table 17.

TABLE 17 Reaction % % % Ex. time, h P—NH₂ P—OH P—OMe 92 2 42.15 0.3 57.55 4 60.73 0.55 38.72 21 99.21 — 0.79

Example 93 Enzymatic Amidation of Z-Ala-Phe-OH

In a typical experiment, the assay mixture contained approximately 10 mMpeptide substrate with a C-terminal free acid and 100 mM ammoniumcarbamate in an anhydrous solvent mixture of 82.5% Bu^(t)OH and 17.5%(vol/vol) of DMF, in a total reaction volume of 5 ml. The reactionmixture was thermostated at 30° C. The reaction was initiated by theaddition of 4 mg/ml Alcalase CLEA. Reaction was incubated at 30° C. for21 hours. Aliquot samples were taken at 2 h, 4 h and 21 h and thereaction was stopped by the addition of an equal volume of acetonitrile.Samples were analyzed by HPLC. The results are listed in Table 18.

TABLE 18 Reaction Z-Ala-Phe- Z-Ala-Phe- Ex. time, h NH₂ a/a % OH a/a %93 2 29.14 70.86 4 34.43 65.57 21 69.86 30.14

It can be concluded from these experiments that the C-terminal free acidof a peptide substrate can also be amidated by ammonium carbamate in thepresence of Alcalase CLEA.

1. A process for the amidation of C-terminal esters or acids of peptidesubstrates in solution-phase synthesis of peptides, comprising amidatingone or more peptide substrates comprising C-terminal esters or acidsusing the protease subtilisin in any suitable form in the presence ofone or more ammonium salts derived from an acid having a pKa above
 0. 2.A process according to claim 1, wherein C-terminal esters of peptidesubstrates are amidated.
 3. A process according to claim 2, wherein theesters of the C-terminal esters of the peptide substrate are selectedfrom the group of C₁₋₁₂ (ar)alkyl esters.
 4. A process according toclaim 3, wherein the esters of the C-terminal esters of the peptidesubstrate are selected from the group of primary C₁₋₁₂ (ar)alkyl esters.5. A process according to claim 4, wherein the esters of the C-terminalesters of the peptide substrate are selected from the group of primaryC₁₋₄ alkyl esters.
 6. A process according to claim 5, wherein the esterof the C-terminal ester of the peptide substrate is the C-terminalmethyl ester.
 7. A process according to claim 1, wherein the ammoniumsalt is derived from an acid having a pKa above 3.5.
 8. A processaccording to claim 7, wherein the ammonium salt has the followingchemical structure (I):

wherein R1 is selected from the group of hydrogen, C₁₋₁₂ (ar)alkyl,C₆₋₁₂ aryl, —N(R2)₂, —OH, and R3-O⁻NH₄ ⁺, R2 is selected from the groupof hydrogen and/or C₁₋₄ alkyl, and R3 is a bond, a carbonyl group, or aC₁₋₄ alkyl carbonyl group, optionally substituted with one or morehydroxyl groups and/or —COO⁻NH₄ ⁺, optionally in hydrated form.
 9. Aprocess according to claim 8, wherein R1 is selected from the group ofC₁₋₁₂ (ar)alkyl, C₆₋₁₂ aryl, —NH₂, —OH, and —O⁻NH₄ ⁺.
 10. A processaccording to claim 9, wherein the ammonium salt is selected fromammonium carbamate, ammonium carbonate, ammonium bicarbonate, ammoniumacetate, ammonium benzoate, and mixtures thereof.
 11. A processaccording to claim 10, wherein the ammonium salt is ammonium carbamate.12. A process according to claim 1, wherein the molar ratio of ammoniumsalt to peptide substrate ranges from 2:1 to 20:1.
 13. The processaccording to claim 1, wherein the peptide substrate comprising theC-terminal ester or acid comprises a C-terminal acyl residue which is anα-amino acyl residue from natural or synthetic origin.
 14. The processaccording to claim 13, wherein the α-amino acyl residue is selected fromAla, protected Cys, protected Asp, protected Glu, Phe, Gly, His,(protected) Lys, Leu, Met, Asn, Gln, (protected) Arg, (protected) Ser,Thr, Val, (protected) Trp and (protected) Tyr.
 15. The process accordingto claim 1, wherein the peptide substrate is prepared according to aprocess for rapid solution synthesis of a peptide in an organic solventor a mixture of organic solvents, the process comprising repetitivecycles of steps (a)-(d): a) a coupling step, using an excess of anactivated carboxylic component to acylate an amino component, b) aquenching step in which a scavenger is used to remove residual activatedcarboxylic functions, wherein the scavenger may also be used fordeprotection of the growing peptide, c) one or more aqueous extractionsand optionally, (d) a separate deprotection step, followed by one ormore aqueous extractions, whereby in at least one cycle in process stepb an amine comprising a free anion or a latent anion is used as ascavenger of residual activated carboxylic functions.
 16. The processaccording to claim 1, wherein the protease subtilisin is of the familyEC 3.4.21.62.
 17. The process according to claim 1, wherein the proteasesubtilisin is free subtilisin.
 18. The process according to claim 1,wherein the protease subtilisin is cross-linked enzyme aggregate (CLEA)subtilisin.
 19. The process according to claim 1, wherein an organicsolvent is used.
 20. The process according to claim 19, wherein theorganic solvent is selected from N,N-dimethylformamide (DMF),N-methyl-2-pyrrolidone (NMP), dioxane, N,N-dimethylacetamide (DMA),dichloromethane (DCM), tetrahydrofuran (THF), acetonitrile,tert-butanol, tert-amyl alcohol, dichloroethane (DCE), tert-butyl methylether (MTBE), and mixtures thereof.
 21. The process according to claim20, wherein the organic solvent is a mixture of tert-butanol and DMF,tert-butanol and NMP, tert-amyl alcohol and DMF, or tert-amyl alcoholand NMP.
 22. The process according to claim 19, wherein water is presentin the organic solvent ranging from 0.0001 to 5% (v/v).
 23. The processaccording to claim 1, wherein pH at which the reaction is performed isselected from the range of 5.5-10.
 24. The process according to claim 1,wherein the reaction temperature for the amidation is 15-60° C.
 25. Theprocess according to claim 1, wherein the amount of protease subtilisinranges from 1 to 50 wt. % related to the peptide substrate.
 26. Theprocess according to claim 1, wherein the amidation is performed bystepwise adding portions of the protease subtilisin (in any suitableform) into the reaction mixture comprising one or more peptidesubstrates comprising C-terminal esters or acids.
 27. The processaccording to claim 1, wherein the amidation is performed by stepwiseadding portions of the ammonium salt into the reaction mixturecomprising one or more peptide substrates comprising C-terminal estersor acids.